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MT 6315 (Cytogenetics) > UNIT 8.2 FISH > Flashcards

UNIT 8.2 FISH Flashcards

1
Q

A cytogenetic technique that uses fluorescent probes that bind specifically to a part of chromosomes complementary to its sequence

A

Fluorescence In-Situ Hybridization (FISH)

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2
Q

Hybridizing two complementary sequences such as

A

probe and target sequence

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3
Q

small sequence of nucleotides that are complementary to a DNA sequence

A

probes

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4
Q

Useful in detecting and mapping the presence or absence of particular DNA sequences within chromosomes

A

Fluorescence In-Situ Hybridization (FISH)

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5
Q

Detects and localizes specific DNA sequences using fluorescently labeled complementary DNA probes.

A

Fluorescence In-Situ Hybridization (FISH)

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6
Q

Detection is through?

A

fluorescence

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7
Q

T OR F

No fluorescence means that no hybridization occurred, therefore the target sequence is present

A

FALSE

target sequence should be absent

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8
Q

Rapid diagnosis of ___ and ___ is acquired using specific probes

A

trisomies and microdeletions

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9
Q

To separate DNA strands and
allow probe access to target
DNA

A

Denature

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10
Q

the process wherein the DNA uncoils and strands separate.

A

Denaturation

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11
Q

Together to bind probe to target
DNA

A

Hybridize

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12
Q

Probe signals using a
fluorescent microscope

A

Analyze

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13
Q

Metaphase or Interphase

Gold standard and routinely done

A

Metaphase FISH

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14
Q

Metaphase or Interphase

Done on cultured cells

A

Metaphase FISH

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15
Q

Metaphase or Interphase

Allows direct visualization of chromosomes and exact position of signals

A

Metaphase FISH

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16
Q

Metaphase or Interphase

useful in the detection of structural changes in the genome; for molecular analysis

A

Metaphase FISH

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17
Q

Metaphase or Interphase

May also be done on
uncultured specimens

A

Interphase FISH

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18
Q

Metaphase or Interphase

Advantageous in the rapid screening of many nuclei for prenatal diagnosis and newborn studies; detection of genetic abnormalities

A

Interphase FISH

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19
Q

Metaphase or Interphase

beneficial in the study of samples with a low mitotic index, such as most solid tumors

A

Interphase FISH

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20
Q

Metaphase or Interphase

Major disadvantage is the inability to detect unknown structural chromosomal changes

A

Interphase FISH

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21
Q

samples for Interphase FISH

A
  1. Amniocytes
  2. Peripheral blood smears
  3. Bone marrow aspirate smear or direct harvest
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22
Q

samples for Metaphase FISH

A
  1. Amniocytes
  2. Chorionic villous cells
  3. Lymphocytes
  4. Cells from bone marrow aspirates or solid tumors fibroblasts
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23
Q

translocation or copy number analysis in cancer studies

A

Bone marrow aspirate smear or direct harvest

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24
Q

For ploidy analysis in newborns

A

Peripheral blood smears

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25
Q

Found within the brain.
Produces CSF

A

Chorionic villous cells

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26
Q

For ploidy analysis during prenatal studies

A

Amniocytes in Interphase FISH

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27
Q

Cells from amniotic fluid

A

Amniocytes in Metaphase FISH

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28
Q

Complementary sequences of target nucleic acids (DNA, RNA or nucleic acid analogs) tagged or labeled with fluorophores

A

FISH Probes

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29
Q

Complementary binding of base pairs

A

(A-T, G-C)

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30
Q

Size ranges from ___ to ___ base pairs

A

20 to 1000

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31
Q

T OR F

The longer the base pairs, the less specific, and the less expensive

A

FALSE

should be more specific and more expensive

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32
Q
  • Fluorophores are directly attached to the probe
  • Less sensitive
A

Direct Labeling

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33
Q

examples of direct labeling

A
  1. Fluorescent isothiocyanate (FITC)
  2. Rhodamine
  3. Cyanines
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34
Q

■ Most commonly used label
■ Apple green signal

A

Fluorescent isothiocyanate (FITC)

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35
Q

Reddish signal

A

Rhodamine

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36
Q

Bluish signal

A

Cyanines

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37
Q

More expensive but is preferred because the materials used add up to the sensitivity of the probe

A

Indirect Labeling

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38
Q

Major disadvantage is that the process is much more tedious due to additional steps

A

Indirect Labeling

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39
Q

Chemical conjugation of the nucleic acid with a nonfluorescent molecule that can bind fluorescent material after hybridization

A

Indirect Labeling

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40
Q

examples of indirect labeling

A
  1. Biotin
  2. Digoxigenin
  3. Antibodies
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40
Q

Commonly used due to having many sources

A

Biotin

41
Q

Biotin is an essential nutrient of

A

hair

42
Q

Biotin is complementary to Avidin which is acquired from ___

A

eggs

43
Q

T OR F

What is bound to the probe is fluorophore and the Biotin is attached to Avidin

A

FALSE

fluorophore = Avidin
probe = Biotin

44
Q

Avidin and Biotin will bind, forming a complex with the ____ attached

A

fluorophore

45
Q

Binds to a particular region of a chromosome

A

Locus Specific Probe

46
Q

Used when only a small portion of a gene is isolated and want to determine in which chromosome the gene is located, or how many copies of a gene exist within a particular genome

A

Locus Specific Probe

47
Q

Single or Dual Color FISH Probe

Designed to cover a gene of interest

A

Single Color FISH Probe

48
Q

Single or Dual Color FISH Probe

Uses one probe and one fluorophore specific to an area

A

Single Color FISH Probe

49
Q

Single or Dual Color FISH Probe

Designed to cover any 2 genes for the detection of any aberrations.

A

Dual Color FISH Probe

50
Q

Single or Dual Color FISH Probe

Allows simultaneous detection of numerical abnormalities of two to three regions in one FISH assay.

A

Dual Color FISH Probe

51
Q

Single or Dual Color FISH Probe

Uses two probes labeled with two different fluorophores

A

Dual Color FISH Probe

52
Q

Generated from repetitive sequences found in the middle of each chromosome

A

Alphoid or Centromeric Repeat

53
Q

Used to determine whether on individual has the correct number of chromosomes or if there is aneuploidy in the patient’s genome

A

Alphoid or Centromeric Repeat

54
Q

Targets centromere and is designed to be complementary to it

A

Alphoid or Centromeric Repeat

55
Q

Specific to the subtelomere region of chromosome

A

Subtelomeric Probe

56
Q

Useful in the detection of subtelomere deletions and rearrangements

A

Subtelomeric Probe

57
Q

If the reason behind down syndrome cannot be identified, this is used.

A

Subtelomeric Probe

58
Q

Collection of smaller probes that bind to the whole length of chromosome

A

Whole Chromosome Probe

59
Q

Smaller probes are specific to an individual locus in a chromosome

A

WHOLE CHROMOSOME PROBE

60
Q

Tag the whole chromo by collecting different small probes, not just a one long probe

A

Whole Chromosome Probe

61
Q

T or F

Whole chromosome probe is useful in the examination of chromosomal aberrations

A

TRUE

62
Q

Specific to chromosomes to determine if the fetus would develop common anueploidies/trisomies

A

Pre-natal FISH Probe

63
Q

Comprised of different combinations of fluorophore-labeled probes specific for chromosomes 13 (Patau), 18 (Edward), 21 (Down), X (Klinefelter), and/or Y (Turner)

A

Pre-natal FISH Probe

64
Q

Types of fish probes

A
  1. alphoid or centromeric repeat
  2. subtelomeric probe
  3. locus specific probe
  4. whole chromosome probe
  5. pre-natal fish probe
65
Q

first step in FISH

A
  1. Probe and target DNAs are denatured using high temperature incubation in a formamide/ salt solution.
66
Q

second step in FISH

A
  1. Probe sequences hybridize to the complementary target sequences, and nonspecific binding is eliminated via stringent washing.
67
Q

most critical step as it removes probes that are unbound to the DNA.

A

Stringent washing (2nd step)

68
Q

T or F (steps in fish)

Probes will continue to fluoresce whether they are bound or unbound.

A

TRUE

69
Q

T or F (steps in fish)

Binding is a signal for fluorescence.

A

FALSE

it is NOT a signal

70
Q

too much / too little results to what?

A

false negative/false positive

71
Q

third step in FISH

A

The probe hybridization is detected with fluorescence microscopy

72
Q

The third or last step in FISH is a fragile procedure because we are dealing with what?

A

small samples such as 5 microliters

73
Q
  • The nucleus should be separate and the boundaries easily seen
  • No overlapping
A

Cell scoring in FISH

74
Q

______ might be due to a twisted nuclei or one on top of another

A

Overlapping

75
Q

A ______ would either be directly next to
each other or have a very small space between them

A

split signal

76
Q

APPLICATION OF FISH

A
  1. Detection and characterization of chromosome
  2. Detection and analysis of prenatal chromosomal abnormalities
  3. Study of chromosomal abnormalities associated with cancer
77
Q

A technique that uses DNA from the cells of interest, rather than using a standard karyotype, for chromosomal analysis.

A

CGH on metaphase cells

78
Q

CGH on metaphase cells can be very useful, especially in some ____ when only DNA is available rather than any growing cells.

A

cancers

79
Q

CGH on metaphase cells has been used successfully for clinical analysis, particularly with cases that have a ____

A

low (or no) mitotic index.

80
Q

CGH on metaphase cells is not useful for detecting _____

A

balanced rearrangements

81
Q

T or F

CGH on metaphase cells has low sensitivity and specificity

A

FALSE

should be high sensitivity

82
Q

A technique that allows the investigator to view a karyotype so that each chromosome is “painted” with a different color

A

Multiplex FISH (M-FISH)

83
Q

MULTIPLEX FISH (M-FISH)

____ are used to create a distinct computer-generated false color for each chromosome

A

Ratio-labeled probes (invisible colors detected by computer)

84
Q

MULTIPLEX FISH (M-FISH)

Useful for complex rearrangements, such as
those seen in ___ and ____

A

neoplastic disorders and solid tumors

85
Q

Uses chromosome-specific mixtures of partial chromosome paints that are labeled with various fluorochromes.

A

Multicolor banding (mBAND) analysis

86
Q

MULTICOLOR BANDING (mBAND) ANALYSIS

A computer program analyzes metaphase chromosome data and produces a ______

A

pseudo colored

87
Q

MULTICOLOR BANDING (mBAND) ANALYSIS

banded karyotype with an estimated resolution of _____, regardless of chromosome length.

A

550 bands

88
Q

Advantageous for the determination of breakpoints and the analysis of intrachromosomal rearrangements, and can be particularly useful in preparations with shorter chromosomes.

A

Multicolor banding (mBAND) analysis

89
Q

Multicolor banding

Region-specific probes labeled with different
_______ and ________ produces a definable number of colored bands per chromosome, regardless of chromosome length.

A

partial chromosome paints (PCP) and computer false color (MetaSystems’ mBAND)

90
Q

A technique that is almost entirely used for research

A

Fiber FISH

91
Q

T or F

fiber fish allows the chromosomes to be stretched out and elongated

A

TRUE

92
Q

This provides a much higher spatial resolution and allows for correct orientation and placement of probes and for precise mapping of probes

A

Fiber FISH

93
Q

Tries to extend chromosome; chromosome stretched to a string and bound with a probe

A

Fiber FISH

94
Q

Essentially PCR on a slide.
All procedures are done on that slide.

A

Primed In Situ Labeling (PRINS)

95
Q

PRIMED IN SITU LABELING (PRINS)

Primers of interest are hybridized on a slide and then subjected to cycles of ___, ____ and _____ that are used to incorporate labeled nucleotides. The labels are
then detected fluorescently or labeled nucleotides are incorporated during the reaction

A
  • denaturation, reannealing, and elongation
96
Q

PRINS can differentiate hybridization with the alpha satellite sequences for chromosomes ___ and ___, something that cannot be done with traditional FISH

A

13 and 21

97
Q

PRIMED IN SITU LABELING (PRINS)

Metaphase chromosomes are subjected to PRINS with _______ specific for chromosomes X, 11, and 17

______ staining is seen at the centromeres of these chromosomes

A
  • alpha satellite oligonucleotides
  • Bright yellow fluorescein
98
Q

Used to identify material of unknown origin

A

Reverse FISH

99
Q

REVERSE FISH

This unidentified material, such as a marker chromosome or duplication, is ____ or _____ of a slide after G-banding. The DNA from this material is extracted, PCR-amplified and labeled with a fluorochrome. This is then used as a probe and hybridized to normal or patient metaphase chromosomes to identify the origin of the unknown material

A

flow sorted or microdissected

100
Q

T or F

in reverse fish, if binding occurs, then the unknown sequence is found

A

TRUE

101
Q

what are the specialized and evolving technologies

A
  • CGH on metaphase cells
  • multiplex FISH (M-FISH)
  • multicolor banding (mBAND) analysis
  • fiber FISH
  • primed in situ labeling (PRINS)
  • reverse FISH

MT 6315 (Cytogenetics) (11 decks)

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